Description

Real Time One Step RNA PCR Kit Ver.2.0 is designed to allow real time RT-PCR reaction in one step with the use of TaqMan^®*1 probe. It can be used with various real time PCR instruments available on the market. As RT-PCR reaction completes in a single tube, the operation is simple, and it minimizes the risk of contamination. Amplified product are monitoring in real time, so there is no need to verify them through eletrophoresis after PCR. This kit is suitable for detection of tiny amount of RNA like RNA virus. This is supplied with ROX™^*2 Reference Dye to nomalize the fluorescent signal between reactions, for instruments that are equipped with this option.

Applicable real time PCR instruments;

• ABI PRISM^® 7000/7700/7900,^*3
• Applied Biosystems 7300/7500 Real Time PCR System (Applied Biosystems)
• LightCycler^® (Roche Diagnostics)^*4
• Smart Cycler^® (Cepheid)^*5

*1 TaqMan^® is a registered trademark of Roche Molecular Systems Inc.
*2 ROX™ is a trademark of Applera Corporation or its subsidiaries in the US and/or certain other countries.
*3 ABI PRISM^® is a registered trademark of Applera Corporation.
*4 LightCycler^® is a registered trademark of Roche Diagnostics.
*5 Smart Cycler^® is a registered trademark of Cepheid.

Reagents and Instruments required but not included in the kit

1. Thermal Cycler for real time PCR
2. Reaction tube or plate for real time PCR
3. PCR primer
4. TaqMan^® probe (licensed probes)
5. dH₂O
6. Micropippets and Micropippet tips (autoclaved prior to use)

Storage

–20°C

Feature

1. Simple protocol and low contamination risk:
   This kit is one-step reaction system and there is no need to add a reagent on the way of reaction. So the protocol is simple and it also minimizes the cross contamination among samples.


2. High sensitivity by preventing non-specific amplification:
   This kit employs TaKaRa Ex Taq™ HS, the most optimal Hot Start enzyme for real time PCR. Accordingly, non-specific amplification derived from mis-priming before thermal cycling or from primer-dimer formation can be prevented.


3. Reaction buffer:

Supplied buffer is optimized to perform both reverse transcription and PCR reaction efficiently in a single buffer composition.

4. Primers:
   Specific primer is necessary for reverse transcription.
   Neither random primer or oligo dT primer can be used.

Kit Components (100 reactions ×25µl PCR)

2 × One Step RNA PCR Buffer*1	630 µl × 2
Reverse Transcriptase XL (AMV)*2 (5 units/µl)	50 µl
RNase Inhibitor (40 units/µl)	50 µl
TaKaRa Ex Taq™ HS (5 units/µl)	50 µl
RNase Free dH2O	1.25 ml × 1
ROX™ Reference Dye ( 50 ×)*3	50 µl
ROX™ Reference Dye II(50 × conc.)*3	50 µl

*1	contains dNTP Mixture, Mg2+
*2	Manufactured by Life Sciences Inc.
*3	ROX™ Reference Dye/Dye II is supplied for performing normalization of fluorescent signal intensities among wells when used with real time PCR instruments that have this option. For ABI PRISM® 7000/7700/7900HT and Applied Biosystems 7300 Real-Time PCR System, the use of ROX™ Reference Dye is recommended. For Applied Biosystems 7500 Real-Time System, the use of ROX™ Reference Dye II is recommended. The use of ROX™ Reference Dye/Dye II is optional, and not required when using Smart Cycler® and LightCycler® real time instrument.